Accepted Manuscript
Title: Assessment of efficacy of a bivalent BTV-2 and BTV-4 inactivated vaccine by vaccination and challenge in cattle Authors: G. Savini, C. Hamers, A. Conte, P. Migliaccio, B.
Bonfini, L. Teodori, M. Di Ventura, P. Hudelet, C.
Schumacher, V. Caporale
PII: S0378-1135(08)00214-9
DOI: doi:10.1016/j.vetmic.2008.05.032
Reference: VETMIC 4062
To appear in: VETMIC Received date: 30-1-2008 Revised date: 20-5-2008 Accepted date: 26-5-2008
Please cite this article as: Savini, G., Hamers, C., Conte, A., Migliaccio, P., Bonfini, B., Teodori, L., Di Ventura, M., Hudelet, P., Schumacher, C., Caporale, V., Assessment of efficacy of a bivalent BTV-2 and BTV-4 inactivated vaccine by vaccination and challenge in cattle,Veterinary Microbiology(2007), doi:10.1016/j.vetmic.2008.05.032 This is a PDF file of an unedited manuscript that has been accepted for publication.
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Accepted Manuscript
ASSESSMENT OF EFFICACY OF A BIVALENT BTV-2 AND BTV-4 INACTIVATED VACCINE 1
BY VACCINATION AND CHALLENGE IN CATTLE 2
3
Savini G1*., Hamers C2., Conte A1., Migliaccio P1., Bonfini B1., Teodori L1., Di Ventura M1. 4
Hudelet P2., Schumacher C2. Caporale V1. 5
1 :Istituto Zooprofilattico Sperimentale dell'Abruzzo e Molise "G. Caporale", Via Campo Boario, 6
64100, Teramo, Italy 7
2 : MERIAL S.A.S. 254 rue M.Mérieux 69007 Lyon France 8
9
Corresponding author: Giovanni Savini 10
Dept. of Virology 11
OIE Reference laboratory for Bluetongue 12
Istituto Zooprofilattico Sperimentale dell’Abruzzo e Molise “G. Caporale”
13
Via Campo Boario, 64100 - Teramo - Italy 14
Telephone: +390861 332440 15
Fax No. +390861 332251 16
E-mail: g.savini@izs.it 17
18
ABSTRACT 19
The efficacy of a bivalent inactivated vaccine against bluetongue virus (BTV) serotypes 2 (BTV- 20
2) and 4 (BTV-4) was evaluated in cattle by general and local examination, serological follow- 21
up, and challenge.
22
Thirty-two 4 month-old calves were randomly allocated into 2 groups of 16 animals each. One 23
group was vaccinated subcutaneously (s/c) with two injections of bivalent inactivated vaccine 24
at a 28-day interval, and the second group was left unvaccinated and used as control. Sixty- 25
five days after first vaccination, 8 vaccinated and 8 unvaccinated calves were s/c challenged 26
with 1 mL of 6.2 Log10 TCID50/mL of an Italian field isolate of BTV serotype 2, while the 27
remaining 8 vaccinated and 8 unvaccinated animals were challenged by 1 mL of 6.2 Log10 28
Manuscript
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TCID50/mL of an Italian field isolate of BTV serotype 4. Three additional calves were included in 29
the study and used as sentinels to confirm that no BTV was circulating locally.
30
At the time of the challenge, only one vaccinated animal did not have neutralizing antibodies 31
against BTV-4, while the remaining 15 showed titres of at least 1:10 for either BTV-2 or BTV-4.
32
However, the BTV-2 component of the inactivated vaccine elicited a stronger immune response 33
in terms of both the number of virus neutralization (VN) positive animals and antibody titres.
34
After challenge, no animal showed signs of disease. Similarly, none of the vaccinated animals 35
developed detectable viraemia while bluetongue virus serotype 2 and 4 titres were detected in 36
the circulating blood of all unvaccinated animals, commencing on day 3 post challenge and 37
lasting 16 days. It is concluded that administration of the bivalent BTV-2 and 4 inactivated 38
vaccine resulted in a complete prevention of detectable viraemia in all calves when challenged 39
with high doses of BTV-2 or BTV-4.
40 41
KEY WORDS: Bluetongue virus serotype 2, Bluetongue virus serotype 4, Cattle, Inactivated 42
vaccine 43
44
INTRODUCTION 45
Bluetongue is an infectious, non-contagious disease of wild and domestic ruminants caused by 46
an RNA virus belonging to the family Reoviridae, genus Orbivirus. Biting midges of the 47
Culicoides genus are the biological vector of the virus, and their distribution affects the 48
spreading of the infection in the temperate and tropical regions of the world (Gibbs et al., 49
1994; Tabachnick, 2004). The disease is typically evident in sheep and only recently clinical 50
cases have unexpectedly been observed in cattle (Verwoerd and Erasmus, 2004; Toussaint et 51
al., 2006). Since 1998, BTV infection has spread progressively all over the Mediterranean 52
Basin, Balkan areas and more recently in Northern Europe. To date, 7 serotypes have been 53
detected in the Mediterranean Basin: BTV-1, BTV-2, BTV-4, BTV-8, BTV-9, BTV-15 and BTV-16 54
(Mellor and Wittmann, 2002; OIE, 2006a,b,c,d,e,f,g,h; Saegerman et al., 2008). In Italy, the 55
first evidence of BTV infection was recorded in Sardinia in August 2000, and since then, 56
numerous outbreaks of BTV serotypes 1, 2, 4, 9 and 16 have been reported impacting most of 57
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the central and southern regions of Italy (Calistri et al., 2004, OIE, 2006f). The incursion of 58
BTV into Europe is having a considerable negative economic impact, partly due to direct losses 59
from death and reduced production in affected livestock but, more importantly, because of the 60
total ban of ruminant trade between BTV-infected and non-infected areas (Calistri et al., 61
2004). Despite the low occurrence of clinical cases in bovines, BTV is one of the 16 diseases 62
formerly ranked as list ‘A’ by the Office International des Epizooties (OIE). As a consequence, 63
Bluetongue affected countries are banned from trading livestock and livestock products. To 64
reduce direct losses due to disease and indirect losses due to the trade embargo caused by 65
virus circulation, European authorities have been undertaking vaccination campaigns according 66
to their individual national policies, the geographic distribution of the incurring BTV 67
serotype(s), and the availability of appropriate vaccines. Prior to 2003, only live vaccines were 68
used. They were the only product available in the market. They are inexpensive and have 69
proven highly effective in preventing bluetongue disease in the areas where they have been 70
used (Savini et al. 2007). However, they could be inadequately attenuated, depress milk 71
production in lactating sheep; and be teratogenic if used in pregnant animals (Savini et al.
72
2004; MacLachlan et al. 1985). Because of their potential to replicate in the organism attaining 73
titres capable of infecting vectors, vaccine viruses have been demonstrated to spread into the 74
environment (Ferrari et al. 2005, Savini et al. 2007) with the potential for reversion to 75
virulence and re-assortment of their genes with those of wild type BTV (Murray et al 1996, 76
Venter et al. 2004, Ferrari et al. 2005, Monaco et al. 2006).
77
With this respect, whole inactivated virus vaccines would represent a safer alternative. Since 78
2005 BTV inactivated vaccines have been in the market and used in vaccination campaigns in 79
Italy, France, Spain and Portugal. Even though most of the animal movements are associated 80
to cattle, the inactivated vaccine launched in the market was for sheep use only.
81
This paper describes the clinical evaluation of a commercial inactivated vaccine containing 82
purified BTV serotypes 2 and 4 in cattle. An experiment was conducted to determine the level 83
of protection induced by the vaccine against a challenge with homologous serotypes.
84 85
MATERIALS AND METHODS 86
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1. Vaccine 87
A bivalent BTV-2/BTV-4 inactivated vaccine produced at an industrial scale by Merial (France) 88
was used in this study. The vaccine is inactivated and adjuvanted with Saponin/Aluminium 89
hydroxide.
90
Both BTV-2 and BTV-4 vaccine strains originated from France (Corsica).
91
Sixteen cattle were vaccinated with 1 mL of the vaccine on day 0 and on day 28. The vaccine 92
was administered s/c on the lateral face of the neck, height centred on the left (day 0) or right 93
(day 28) side. Rectal temperatures of all animals (including controls) were recorded prior to 94
vaccinations.
95 96
2. Challenge strain 97
Field isolates recovered from Sardinian infected animals during the BTV outbreaks of 2000- 98
2003 were used as challenge strains: the 8341/00 strain for BTV-2 and 10353/03 for BTV-4.
99
Before challenging, each strain was amplified by one passage in VERO (African green monkey 100
kidney) cell cultures. The strains were titrated after cell passage and then used as challenge 101
strain. Viral suspensions were diluted in phosphate buffer saline (PBS, pH 7,2) to adjust the 102
viral titre to 106.2 TCID50./mL. Each animal was challenged s/c on the left face of the neck on 103
day 65 with 1 mL of its respective viral suspension.
104 105
3. Animals 106
This study was conducted between November 2005 and April 2006 in the province of Teramo 107
at an altitude of 1000 m in an insect-proof facility.
108
Thirty-five 4 month-old calves that were free of respiratory, digestive, umbilical and osteo- 109
articular disease were included in the study. All animals originated from BTV-free herd, located 110
in a BTV-free area (France) and tested negative for BTV-antibodies (c-ELISA) before entering 111
the study.
112
Thirty-two calves were randomly allocated into 2 groups of 16 animals each. One group was 113
vaccinated and the second group was left unvaccinated and used as control.
114
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Sixty-five days after first vaccination, 8 vaccinated and 8 unvaccinated calves were challenged 115
by BTV serotype 2, while the remaining 8 vaccinated and 8 unvaccinated animals were 116
challenged by BTV serotype 4.
117
Thus, animals were allocated to four groups : (1) “Controls BTV-2”, (2) “Controls BTV-4”, (3) 118
“Vaccinates BTV-2”, (4) “Vaccinates BTV-4”.
119
The three remaining animals were kept as environmental controls (not vaccinated nor 120
challenged), and used as sentinels to confirm that no BTV was circulating locally.
121
On days 2, 14, 28 (before vaccination), 42, 65 (before challenge), and 86 all cattle were blood 122
sampled by jugular puncture with plain tubes; serum samples were tested by c-ELISA and 123
titrated for antibodies against BTV-2, BTV-4, BTV-9 and BTV-16 by virus neutralisation test 124
(VN).
125
Prior to challenge, all bovines were blood sampled for detection of viraemia by jugular 126
puncture with ethylene-diaminetetra-acetic acid (EDTA) tubes. After challenge, blood samples 127
were taken 3 times a week for 42 days (day 65 -> day 107).
128
To exclude viral circulation in the stable, the environmental control animals were bled once a 129
week for the entire trial length. Serum samples were tested by ELISA. In case of positive 130
reaction they were subsequently typed by VN.
131 132
4. Virological and serological tests 133
The presence and titre of the virus in the blood were assessed by titration on VERO cells, 134
according to the methods described by Savini et al., 2004 and OIE Manual of diagnostic tests 135
and vaccines for terrestrial animals (2004). The presence of BTV in EDTA-blood samples was 136
also assessed by using the RT-PCR which amplifies a portion of S5 as described by Katz et al.
137
(1993). Its sensitivity for BTV-2 and BTV-4 of either field or vaccine origin was evaluated by 138
testing viral suspensions whose titer was previously determined.
139
On day 7, in the absence of cytopathic effect (CPE), cultures were scraped, centrifuged, and 140
the supernatant was re-passaged into a 24 microplate flat bottomed wells containing a 141
confluent monolayer of VERO cells and the plates handled as described before. In the presence 142
of CPE as well as at the end of the second passage, cells were scraped and collected from each 143
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well. Cells and medium were then centrifuged at 812 g for 10 min. and the pellet was re- 144
suspended in approximately 1 mL of PBS. Ten µL of the cell suspension were subsequently put 145
onto a well of a multiwell slide, fixed for 20 min. in acetone at –20° C and checked for the 146
presence of BTV by immunofluorescence (IF) using BTV monoclonal antibodies. Virus 147
characterised as BTV was consequently typed by virus microneutralisation assays using type- 148
specific antisera. The virus titer was determined using the Reed and Munch formula applied on 149
the four replicates after the first passage. Those samples which showed CPE after the second 150
passage or were positive to IF were considered positive for BT with a titer of less than 102.3 151
TCID50/mL. (Savini et al. 2007) 152
As for the virus neutralisation assays, the method described by Savini et al. 2007 was applied 153
to both challenge inocula to demonstrate their serotype. The positive and negative controls as 154
well as OIE standard reference BTV serotypes 2, 4, 9 and 16 were kindly provided by the OIE 155
reference laboratory, Onderstepoort Veterinary Institute (OVI) in South Africa.
156
The antibody response was monitored using both the c-ELISA (Lelli et al., 2003) and VN test 157
(Savini et al., 2004).
158 159
5. Statistical analysis 160
Differences between the neutralising titres against BTV-2 and 4 per sampling day in the 161
vaccinated group after immunisation, were analysed using the non-parametric Wilcoxon test 162
for paired groups (Siegel and Castellani, 1998), while differences between BTV-2 and 4 163
viraemic titres per sampling day in the control groups were analysed using the non-parametric 164
Mann-Whitney test for independent groups. The probability of the various possible sensitivity 165
values of the C-ELISA in the vaccinated animals were estimated through a Bayesian approach 166
using the Beta (s+1, n-s+1) distribution (Sivia, 1996) where s is the total number of positives 167
and n is the total number of tested animals. Different Beta distributions were calculated on the 168
basis of different periods from vaccination. The probability distribution of the percentage of 169
positive animals shows not only the most probable value of sensitivity, but also the level of 170
uncertainty due to sample size. From an epidemiological point of view, it is far more interesting 171
to know the percentage of vaccinated animals that are protected after being challenged with 172
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homologous BTV serotypes. The probability that more than a certain percentage of animals 173
would be protected after challenge was calculated by using 1-Beta (s+1, n-s+1).
174 175
RESULTS 176
1. Virological results 177
After challenge, none of the vaccinated animals developed detectable viraemia by either cell 178
culture or RT-PCR. Conversely bluetongue virus nucleic acid and serotype 2 and 4 titres were 179
detected in the circulating blood of all unvaccinated animals. In these animals the RT-PCR 180
detected BTV starting on day 5 post infection (pi) and remained positive up to the end of the 181
experimental period (42 days pi). The cell culture virus isolation detected BTV-2 and BTV-4 182
titers commencing on day 3 pi and lasting 16 days. In both infections, maximum viral titre was 183
detected on day 10 pi with average titres of 104.02 TCID50/mL and 103.54 TCID50/mL for BTV-2 184
and BTV-4, respectively (Fig. 1). The difference between BTV-2 and BTV-4 viraemic titres was 185
statistically significant (p<0.05), with BTV-2 titres much higher than those with BTV-4. Figure 186
2 shows the curve of the probability that vaccinated animals are protected, not showing any 187
detectable viraemia after challenge infection with homologous BTV.
188 189
2. Serological results 190
None of the vaccinated animals showed detectable c-ELISA antibodies after the first vaccine 191
injection. Conversely, commencing on day 42 (14 days after the second shot), the immuno- 192
enzymatic assay detected antibodies in all vaccinated calves. Figure 3 shows the probability 193
distributions of the percentage of calves showing c-ELISA antibodies after being vaccinated 194
with the inactivated bivalent BTV-2 and 4 vaccine. Apart from those used as environmental 195
controls, all animals were positive to c-ELISA on day 86.
196
For BTV-2, 28 days after first vaccination, 13 had sero-converted, but at low titres. By day 42, 197
all had BTV-2 neutralizing antibodies. For BTV-4, none of the calves had detectable BTV-4 198
neutralizing antibodies 28 days after the first vaccination. By day 42, fifteen out of sixteen had 199
BTV-4 neutralizing antibodies. On day 28, the number of BTV-2 positive calves was 200
significantly higher (p<0.05) than that of BTV-4. In one animal BTV-4 antibodies were not 201
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detected. The difference between BTV-2 and BTV-4 neutralising titres was statistically 202
significant (p<0.05) at all time points between day 14 and day 65, with BTV-2 titres being 203
much higher than those of BTV-4. However, for both serotypes, the highest peakof antibody 204
response was observed on day 42 (Fig. 4). No animals developed BTV-9 and BTV-16 antibody 205
titres. On day 86, BTV-2 and BTV-4 challenged groups developed homologous neutralising 206
antibodies while no BTV antibody titres were observed in the animals used as environmental 207
controls.
208
Neither BTV nor antibodies were detected in the blood samples taken from the environmental 209
control group.
210 211
DISCUSSION 212
Vaccination against BTV is a very important tool, not only for the control of the disease but 213
more importantly, for ‘safe’ trade of live ruminants in accordance to OIE standards and EU 214
legislation. To prevent BTV infection of ruminants, different types of vaccines, including 215
inactivated and modified live virus (MLV) vaccines, Virus-like particles (VLP) produced from 216
recombinant baculoviruses, and recombinant vaccinia virus vectored vaccines have been 217
manufactured (Roy and Erasmus, 1992, Boone et al., 2007).
218
Of these, the inactivated and MLV vaccines only are now available in the market and used in 219
the official vaccination campaigns. Although VLPs are safe and neat, their inconsistent efficacy 220
when used in field trials (Roy et al., 1990, 1992, 1994; Roy, 2004) and difficulties with 221
commercial production, cost, and long-term stability make them ineligible for field use (Savini 222
et al., 2007). Similarly, recombinant vector vaccines expressing both VP2 and VP5, even 223
though revealing some potential in terms of safety and protection, still require further 224
development before being ready for field use (Lobato et al., 1997). Conversely, MLV are 225
cheap, easy to produce in large quantities, able to elicit protective immunity after a single 226
inoculation, and have been proven effective in preventing clinical bluetongue (BT) disease in 227
the areas where they have been used (Patta et al., 2004; Dungu et al., 2004). Nevertheless 228
BTV MLV vaccines suffer from a variety of documented potential drawbacks including side 229
effects due to under-attenuation of the modified strains and their capacity of passing the 230
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placental barrier, and the spread of vaccine strain in the environment with the potential for 231
reversion to virulence and re-assortment with field isolates.
232
Inactivated vaccines have been recently developed and marketed. The efficacy of an 233
inactivated vaccine is fully dependent on the dose of virus, resulting in significantly higher 234
virus mass than that of MLV. Two doses, in the presence of adjuvant, may often be required 235
for inactivated vaccines considerably increasing the cost of vaccination. Inactivated vaccines 236
for BTV-2 and/or BTV-4 have been developed, commercialised and successfully employed in 237
the 2005-2006 BTV vaccination campaigns. Despite this success, the real costs of bluetongue 238
infection came from the ban on trading live animals in general and cattle in particular. The 239
inactivated vaccines available in the market are primarily registered for sheep and very few 240
information were available on the use of these products in cattle, at the time of this study. In 241
this study, the administration of the bivalent BTV-2 and BTV-4 inactivated vaccine was safe 242
and resulted in a complete prevention of detectable viraemia in all calves when infected with 243
high doses of virulent BTV-2 or BTV-4. It occurred when using either the cell culture method or 244
the RT-PCR assay. It has to be said however that, amongst the two assays, the cell culture 245
isolation is the method capable of giving information on the presence of infectious virus. Two 246
doses of the immunological product completely prevented vaccinated animals from developing 247
viraemia, which statistically signify a virological protection of at least 83.8% (95% confidence 248
interval) of vaccinated animals. It means that the product is not only safe but also effective in 249
protecting all vaccinated animals from viraemia, including the calf which did not develop 250
neutralising antibodies against BTV-4. At the time of the challenge, that calf was the only 251
vaccinated animal which did not develop neutralizing antibodies, while the remaining 15 252
showed titres of at least 1:10 for either BTV-2 or BTV-4. The study showed that the BTV-2 253
component of the inactivated vaccine elicited a stronger immune response in terms of both the 254
number of VN positive animals and antibody titres. However, the differences observed might 255
also relate to the VN technique and further studies are required to demonstrate higher 256
immunological stimulation of certain serotypes as compared to others. Concerning the 257
diagnostic tests, it was surprising to note that 13 animals, even if at low titres, had sero- 258
converted for BTV-2 28 days after first vaccination, whereas no c-ELISA antibodies were 259
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detected in vaccinated animals following the first injection. These data were unexpected as c- 260
ELISA normally detects antibodies earlier than VN. It is known that neutralising antibodies are 261
stimulated by the structural proteins of the outer capsid, VP2 and partially VP5, while 262
antibodies to the inside capsid, VP7, are detected by the c-ELISA (Huismans and Erasmus, 1981, 263
Jeggo et al., 1991). An ineffective antigen stimulation by the VP7 following vaccination could be 264
an explanation of this discrepancy, or alternatively, it could be due to a poor performance of 265
the c-ELISA in detecting antibodies in vaccinated animals.
266
It is concluded that the BTV-2 & BTV-4 bivalent inactivated vaccine tested in the experiment 267
safely and effectively induces protective immunity in cattle, making it suitable for BTV 268
vaccination campaigns and particularly ideal for use in BTV-free countries where emergency 269
ring vaccination may be necessary. Moreover, because it is an inactivated vaccine, its use does 270
not prevent the possibility of using DIVA strategy based on the detection of antibodies versus 271
BTV non structural proteins to distinguish vaccinated from infected animals.
272 273
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Accepted Manuscript
Figure 1: Titers of infectious BTV in blood samples of vaccinated and control groups following challenge infection with BTV-2 and BTV-4 field isolates
0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50 4,00 4,50 5,00
-2 3 5 7 10 12 14 17 19 21 24 26 28 31 33 35 38 40 42
Days after challenge Viraemia (log10TCID50/ml)
BTV-2 vaccinates BTV-2 controls BTV-4 controls BTV-4 vaccinates
Figures 1-4
Accepted Manuscript
Figure 2: Probability that calves vaccinated with an inactivated bivalent BTV-2 and BTV-4 vaccine are protected against homologous challenge. The P value is equal or bigger to the x-axis
percentages. According to the trial, at least 83.8% (blue circle) of vaccinated animals with 95%
confidence level would be protected (no viraemia) when challenged with BTV-2 or BTV-4 field isolates.
0,00 0,10 0,20 0,30 0,40 0,50 0,60 0,70 0,80 0,90 1,00
0% 20% 40% 60% 80% 100%
Percentage of protected animals
Probability (X>x)
Accepted Manuscript
Figure 3: Probability distributions of the percentage of c-ELISA positive calves after vaccination with inactivated bivalent BTV-2 and BTV-4 vaccine
0,000 0,001 0,002 0,003 0,004 0,005 0,006 0,007 0,008 0,009 0,010
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Percentage of positive animals
Confidence
up to 28 days post vaccination from 42 days post vaccination
Accepted Manuscript
Figure 4: Average evolution of BTV-2 and BTV-4 serum neutralising titres in the vaccinated and unvaccinated groups
0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 10,0
-2 14 28 42 56 65 86 100
Days after vaccination
Virus neutralising titres (Log2(ED50%/50ml))
BTV-2 vacc BTV-2 cont BTV-4 vacc BTV-4 cont
